Non-heme iron-dependent enzymes (NHIs) offer alluring means to C–H activation reactions and have demonstrated the capability to react with non-native substrates. Bioinformatic tools in tandem with high-throughput reaction screening approaches have enabled the exploration of previously undocumented biocatalysts. This work aims to understand the extent of which non-heme iron enzymes can perform C–H oxidation across a library of NHIs on diverse non-native coumarin scaffolds. A whole cell E.coli library of over 300 alpha-ketoglutarate dependent NHIs was generated, representing biocatalysts across diverse organisms, biosynthetic pathways, and native substrates. Whole cell reactions were screened in 96-well format across a substrate panel of coumarin, 2-pyrone, and 4-pyrone substrates with negative controls. Reactions were pooled and monitored for hydroxylation mass by LCMS, followed by validation and site-selectivity determined for select reactions. Substrates with promising compatibility with chemoenzymatic systems were optimized and elaborated towards the total synthesis of biologically relevant complex molecules. The development of a chemoenzymatic method employing NHIs could offer a facile route under ambient, aqueous conditions for the selective oxidation of coumarins towards drug-like molecules. Thus, NHI enzymes with promising conversion on select coumarins were performed on preparative scale to demonstrate the feasibility of these biocatalysts with functionally diverse coumarins and pyrones. Identifying non-native, novel oxidation reactivity of coumarins with previously undocumented non-heme biocatalysts, this work adds to a growing understanding of how enzyme substrate promiscuity in chemical space is genetically encoded in sequence space. Further, the characterization and chemoenzymatic synthesis of oxygenated coumarins scaffolds mapping onto biologically relevant complex molecules can inform chemoenzymatic total synthesis efforts. This investigation expands the work on non-heme iron-dependent enzymes as biocatalysts for selective C–H functionalization on broad substrate classes.

Non-native C–H oxidation of diverse coumarin scaffolds by non-heme iron enzymes

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